Interaction of enzymes with surfactants in aqueous solution and in water-in-oil microemulsions

Abstract

Kinetic studies have been carried out on hydrolysis reactions catalysed by a variety of lipases and α-chymotrypsin in aqueous solution in the presence of surfactants in order to provide information on the role of the surfactant in similar processes in microemulsions. The main conclusions concerning the influence of surfactants on the activity of lipase and α-chymotrypsin are as follows. For ex-Candida and Chromobacterium viscosum lipase, the kinetic behaviour of the enzyme in the hydrolysis of p-nitrophenylbutyrate in the presence of surfactants was dependent on the source of lipase. Most experiments were carried out with an unfractionated sample of C. viscosum-lipase. Both competitive and non-competitive inhibition was detected, the former with sodium dodecylsulphate (SDS) and cetyltrimethylammonium bromide (CTAB) and the latter with aerosol-OT (AOT). In all cases inhibition was reversible. In addition, lipases are resistant to slow denaturation by surfactants over periods of several hours. For lipases, there is apparently no correlation between the behaviour of the enzyme in aqueous solutions of surfactant and the behaviour in water-in-oil microemulsions stabilised by AOT and CTAB.In contrast, for the hydrolysis of N-glutaryl-L-phenylalanine-p-nitroanilide (GPNA) by α-chymotrypsin in aqueous solution and in microemulsions, we find both inhibiting and denaturing interactions. AOT is a competitive inhibitor for α-chymotrypsin in aqueous solution but enzyme stability is unaffected. The same behaviour is observed in AOT-stabilised microemulsions. The turnover number (kcat) is essentially unchanged in aqueous solution and microemulsions, but the Michaelis constant (KM), expressed as an aqueous concentration, is increased in AOT microemulsions by a factor of ca. 100 for the hydrolysis of GPNA. For C12E6 and SDS we find both inhibition and denaturation interactions with α-chymotrypsin in water. The stability of α-chymotrypsin is decreased in the presence of SDS and C12E6 in water and in microemulsions as compared with systems containing AOT. CTAB does not inhibit α-chymotrypsin (in terms of an increased KM), but CTAB is an effective denaturing agent for α-chymotrypsin. However, the denaturation reaction does not occur in the presence of substrate.The various surfactants form stable inclusion complexes with α-cyclodextrin (α-CD). Hence α-CD is an effective scavenger for the surfactant in solution. On addition of α-CD, the enzyme activity can be restored to that found in the absence of surfactant.